Sep 09, 2022

Immunocytochemistry (ICC)

DOI
https://dx.doi.org/10.17504/protocols.io.q26g74w79gwz/v1
  • 1Queen Square UCL Institute of Neurology;
  • 2The Francis Crick Institute
Minee L Choi
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g74w79gwz/v1
Protocol Citation: mineechoi 2022. Immunocytochemistry (ICC). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74w79gwz/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 25, 2022
Last Modified: May 31, 2024
Protocol  Integer ID: 63207
Keywords: ASAPCRN, immunocytochemistry, immunocytostochemistry, icc, derived cell, cell, protocol
Abstract
This protocol describes how to do immunocytostochemistry for primary and hiPSC-derived cells.
Cell fixation
Cells are fixed in 4 % volume paraformaldehyde (PFA) and stored in phosphate-buffered saline (PBS) until use.





Permeabilizing and blocking cells
Wash cells with PBS twice.
Incubate the cells in 0.2 % volume Triton X-100, 5 % volume bovine serum albumin (BSA) for 01:00:00 atRoom temperature .
Note
5 % volume BSA (made in PBS) is used to block non-specific binding.



Note
For ATTO 425 labelled Aptamer staining, cells are permeabilized with 0.25 % volume Triton X-100 and blocked with 10 % volume normal goat serum (NGS) for 00:20:00 followed by another 03:00:00 with 0.1 % volume Trion X-100 and 10 % volume NGS.




1h
Incubate cells in primary antibodies

Note
Do not wash after permeabilising and blocking steps

Dilute primary antibody in 5 % volume BSA and incubate cells at 4 °C , Overnight or 01:00:00 at Room temperature .
Note
The final volume should be sufficient to cover each coverslip around 170 µL for 8-ibid chambers, #80806). For 8-ibid, it recommends incubating the cells at room temperature for 01:00:00 at Room temperature if possible.


2h
Wash cells with 5 % volume BSA for 00:05:00 three times.

5m
Incubate cells in secondary antibodies
1h 5m
Dilute primary antibody in 5 % volume BSA and incubate cells at 4 °C Overnight or 01:00:00 at Room temperature .

1h 5m
Wash cells with 5 % volume BSA for 00:05:00 three times away from light.
Note
Add Hoechst (10 micromolar (µM) ) in the second wash and leave for 00:15:00 .



5m
Take away PBS and load anti-fading medium to cover cells.
Note
For the short term, imaging in PBS is also fine.